BACKGROUND: Infective endocarditis (IE) is a life-threatening disease with high mortality, particularly when caused by Staphylococcus aureus (S aureus). Traditional diagnosis relies on clinical assessment, blood cultures, and imaging (eg, echocardiography) but lacks methods for direct bacterial detection on the endothelium. Positron emission tomography (PET)/computed tomography is emerging as valuable in unclear IE cases. This study evaluates 2-[18F]F-p-aminobenzoic acid (2-[18F]F-PABA), a bacteria-specific PET tracer, for detecting IE. METHODS: In vitro assays measured 2-[18F]F-PABA uptake by S aureus, distinguishing living bacteria from dead cells. A mouse model of IE provided proof-of-concept PET/magnetic resonance imaging (MRI) data for diagnosing IE. Additionally, a subcutaneous abscess model with larger bacterial vegetations was used to enhance imaging of bacterial uptake. RESULTS: 2-[18F]F-PABA uptake by S aureus was confirmed in vitro, with accumulation limited to living bacteria and varying across strains. In vivo, 2-[18F]F-PABA PET/MRI successfully visualized IE in mice. In the subcutaneous abscess model, the tracer effectively localized S aureus vegetations, emphasizing its potential in detecting bacterial foci. CONCLUSIONS: This study highlights the potential of 2-[18F]F-PABA PET imaging for direct visualization of IE. Future research may further investigate its diagnostic performance in larger models, aiming toward clinical application as a precise, pathogen-specific imaging tool for managing IE.